Immune therapy involves the exposure of components of the immune system to various elements (cytokines, disease associated antigens and natural metabolites) to combat disease processes in which a dysregulated immune response is thought to play a role. Immune dysregulation is thought to play a major part in the pathogenesis or disease course of a great number of disease processes, including various neoplastic, inflammatory, infectious and genetic entities.
These disorders can be perceived as a dysbalance between pro-inflammatory (Th1) and anti-inflammatory (Th2) cytokines, and few of them are described herein below.
The role of the Immune System in the Pathogenesis of Inflammatory Bowel Disease
Inflammatory bowel diseases (IBD) are common gastrointestinal disorders, that can be perceived as being the result of a dysbalance between Th1-pro-inflammatory, and Th2-anti-inflammatory subtypes of immune responses [Strober, W., et al., Immunol Today 18:61-64 (1997); Neurath, M., et al., J. Exp. Med. 183:2605-2616 (1996)].
There are several extra-intestinal manifestations that accompany IBD, for example: autoimmune phenomena; immune complexes have a role in target organ damage; and, immunosuppressive agents such as glucocorticoids, azathioprine, methotrexate and cyclosporin are used to alleviate the disease [Podolsky, D. K., et al., New Engl. J. Med., 325:928-935(1991); Strober, W., et al., In Clinical Immunology, Mosby, St. Louis. R. R. Rich, Editor, 1401-14281-2 (1995)]. Patients with IBD have antibodies against components of colon cells and several different bacterial antigens. These antigens gain access to the immune system as a consequence of epithelial damage [Hibi, S., et al., Clin. Exp. Immunol. 54:163-168 (1983); Das, K. M., et al., Gastroenterology 98:464-69 (1990)]. Abnormalities of T cell-mediated immunity, including coetaneous anergy and diminished responsiveness to T cell stimuli, have also been described in these patients [Chiba, M., et al. Gut, 22:177-182 (1981); Raedler, A., et al., Clin. Exp. Immunol. 60:518-526 (1985)]. In addition, changes in mucosal cell mediated immunity were identified, including increased concentrations of mucosal IgG cells and changes in T cells subsets, suggesting antigen stimulation [Dasgupta, A., et al., Gut 35:1712-17 (1994); Takahashi, F., et al., J. Clin. Invest. 76:311-318 (1985)]. Exposure of target antigens after infectious, immune, or toxic damage, leads to activation of mucosal immune cells resulting in cytokines that lead to mucosal inflammatory response [Neurath, M., et al., J. Exp. Med., 183:2605-2616 (1996)]. Secretion of pro-inflammatory cytokines such as IFNγ, contributes to an increase in mucosal permeability, and has been described in animal models of IBD [Strober, W., et al., Immunol. Today 18:61-64. (1997)]. Similarly, an increase in collagen synthesis mediated by IL1 and IL6 can be detected in these animals [Strober, W., et al., ibid.]. A Th1-mediated granulomatous colitis model has been established by the adoptive transfer of normal CD45RB T cells from Balb/C mice into CB-17 scid mice. CD4 cells from CD45RB were shown to prevent the disease when injected together with the CD45RB population. This prevention could be reversed by adding antibodies to TGFβ1 [Sadlack, B., et al., Cell 75:253-261 (1993); Powrie, F., et al., Immunity 1:553-562 (1994)].
The Th1/Th2 Dysbalance in Inflammatory Bowel Disease
Both CD4 and CD8 lymphocytes can be typed as either Th1 cells that produce IL-2 and IFNγ, or Th2 cells that produce IL-4, and IL-10. The way the immune system responds to foreign and self antigens, is the result of a balance between the two subtypes of responses [Weiner, H. L., et al., Immunol. Today 18: 335-343 (1997); Adorini, L., et al., Immunol. Today 18:209-211 (1997)]. A Th1 type response is involved in the pathogenesis of several autoimmune and chronic inflammatory disorders such as IBD [Adorini, L., et al., (1997) ibid.; Mizoguchi, A., et al., J. Exp. Med. 183:847-856, (1996)]. Thus experimental colitis and IBD in humans can be perceived as a dysbalance between pro-inflammatory Th1-type and anti-inflammatory Th2-type cytokines. It has been recently shown, in both animals and humans, that anti-inflammatory cytokines such as IL10 can downregulate the pro-inflammatory effects of Th1-mediated cytokines, thereby alleviating immune-mediated disorders [Mizoguchi, A., et al., (1996) ibid.; Madsen, K. L., et al., Gastroenterology 113:151-159 (1997); Van Deventer Sander, J., et al., Gastroenterology 113:383-389 (1997)].
The Role of the Immune System in the Pathogenesis of Non-Alcoholic Steatohepatitis
Non-alcoholic steatohepatitis (NASH) is a clinico-pathological entity consisting of hepatic fat accumulation, inflammation and fibrosis in patients who have no history of alcohol consumption. It may progress to cirrhosis in 20% of cases and is considered the most common cause of cryptogenic cirrhosis in the Western world [Caldwell, S. H. et al., Hepatology 29:664 (1999); Matteoni, C. A. et al., Gastroenterology 116:1413 (1999)]. NASH is common in patients who suffer of other metabolic disturbances, which are suggested to play a contributing role in the pathogenesis of the disorder. These include insulin resistance [Sanyal, A. J. et al., Gastroenterology 120:1183 (2001)], obesity-related ATP depletion [Cortez-Pinto, H. et al., Jama 282:1659 (1999)], increased free-fatty-acid beta peroxidation [Hruszkewycz, A. M. Biochem. Biophys. Res. Commun. 153:191 (1988)], iron accumulation [George, D. K. et al., gastroenterology 114:311 (1998)], antioxidant depletion [Harrison, S. A. et al., gastroenterology 123:M1332 (2002)], and leptin deficiency [Cohen, B. et al., Science 274:1185 (1996)]. Yet no therapeutic intervention, including weight loss, tight diabetic control, normalization of lipid levels and antioxidant treatment have consistently shown an alteration in the natural progression of the disorder [Angulo, P. New England Journal of Medicine 346:1221-1231 (2002)].
Most information about NASH has been derived from two mammalian models: leptin-deficient ob/ob mice and leptin-receptor deficient fa/fa Zucker rats. Leptin is a protein that is involved with the regulation of body weight [Zhang, Y. et al., Nature 372:425-432 (1994)]. Its deficiency in rodents and humans results in a severe form of ‘metabolic syndrome’ (formerly termed syndrome X) consisting of morbid obesity, glucose intolerance, hyperlipidemia, and severe hepatic steatosis [Pelleymounter, M. A. et al., Science 269:540-543 (1995)]. Yet, as mentioned above, no intervention aimed at correcting some of these metabolic disturbances have resulted in an amelioration of the hepatic steatosis, fibrosis, and inflammation.
Recent evidence suggests that the immune system may play a pivotal role in the pathogenesis of NASH in the leptin deficient models. In leptin deficient mice, defective hepatic macrophage (Kupffer cell) response has been observed after liver injury induction by lipopolysaccharide [Diehl, A. M. J. Physiol. Gastrointest. liver Physiol. 282:G1-G5 (2002)]. In similar models, LPS induction of IL6 was greatly enhanced, while that of IL10 was inhibited [Loffreda, S, et al., FASEB J. 12:57-65 (1998)]. Ob/ob mice hepatic macrophages were observed to produce more IL12 and less IL15 than control mice in response to LPS challenge, which may explain the significant reduction in the number and function of NKT lymphocytes observed in these mice [Yang et al., Proc Natl Acad Sci USA 94:2557-2562 (1997)]. Other observations have shown a reduction in the number of CD4 T lymphocytes in the blood and liver of leptin-deficient ob/ob mice [Howard, J. K. et al, J. Clin. Invest. 104:1051-1059 (1999) and Lord, et al., Nature 394:897-901 (1998)]. This may explain the relative resistance of leptin-deficient mice to Concanavalin A hepatitis, which is mediated by CD4 T lymphocytes [Faggioni, R. et al., Proc. Natl. Acad. Sci. USA 97:2367-2372 (2000)].
The Th1/Th2 Dysbalance in Non-Alcoholic Steatohepatitis
CD4 and CD8 lymphocytes are classified as either Th1 cells that produce IL-2 and IFNγ, or Th2 cells that produce IL-4 and IL-10. The immune system responds to foreign and self-antigens by a shift in balance between the two subtypes of responses [Weiner, H. L. et al., Immunol. Today 18: 335-343 (1997); Adorini, L. et al., Immunol. Today 18:209-211 (1997)]. Usually the Th1 type response causes a pro-inflammatory reaction [Adorini, L. et al., (1997) ibid.; Mizoguchi, A., et al., J. Exp. Med. 183:847-856, (1996)], while anti-inflammatory cytokines such as IL10 shift the balance towards an anti-inflammatory Th2 reaction, thereby alleviating immune-mediated disorders [Mizoguchi, A. et al., (1996) ibid.; Madsen, K. L. et al., Gastroenterology 113:151-159 (1997); Van Deventer Sander, J. et al., Gastroenterology 113:383-389 (1997)]. NKT cells, in response to different endogenous and exogenous stimuli, are believed to play a major role in the direction of the immune system towards either the Th1 or Th2 pathways.
Leptin has been shown to play a major role in the immune regulation of the balance between Th1 & Th2 response (Lord, G. M. et al., Nature 394:897-901 (1998)]. In the leptin-deficient ob/ob mice NASH model an alteration of the number and function of NKT cells has been suggested to tilt the immune system towards the Th1 response. This is suggested to result in an increased sensitivity to LPS induced hepatotoxicity and a unique resistance to the hepatotoxic effects of Concanavalin A. The difference may be in their different pathogenic mechanisms. The former depends upon the action of the innate hepatic immune system, which is hyperactive in the leptin-deficient mice, while the latter is dependent upon the activation of NKT-lymphocytes, which are suppressed and defective in the leptin deficient mice [Faggioni, R. et al., PNAS 97:2367-2372 (2000), Zhiping, L. I. et al., Gastroenterology 123:1304-1310 (2002)].
The Immune System and Obesity
The immune system and the regulation of adipose tissue metabolism appear to be closely interlinked. Up to fifty percent of cells within adipose tissues are composed of non-adipose cells, including many immunocytes [Montague, C. T. et al., Diabetes 47:1384-91 (1998)]. Most research has been focused on the immunological consequences of morbid obesity. Immunological alterations which are known to exist in obese animals and humans include reduced DTH and mitogen-stimulated lymphocyte proliferation responses [Chandra, R. K. et al., Acta. Paediatr. Scand 69:25-30 (1980)], impaired phagocyte number and function [Krishnan, E. C. et al., J. Surg. Res. 33:89-97 (1982)], attenuation of insulin induced lymphocyte cytotoxicity [Koffler, M. et al., Diabetes 40:364-360 (1991)], and changes in the CD4/CD8 ratio, especially during weight loss attempts [Field, C. J. et al., Am. J. Clin. Nutr. 54:123-129 (1991)].
Adipose cells are known to secrete pro-inflammatory cytokines including TNF-β [Hotamisligil, G. S. et al., Science 259:87-91 (1993)] and IL6 [Purohit, A. et al., Journal of Clinical Endocrinology and Metabolism 80:3052-58 (1995)], which are both related to the level of adiposity [Hotamisligil, G. S. et al., Journal of Internal Medicine 245:621-625 (1999)]. Some of these cytokines are considered to have metabolic effects such as insulin resistance mediated by TNF-β [Ogawa, H. et al., Biochimica et biophysica acta 1003:131-135 (1989)] and lipoprotein lipase inhibition mediated by IL6 [Feingold, et al., Diabetes 41:97s-101s (1992)]. TNF-β knockout mice have higher insulin sensitivity and improved lipid profile than their normal littermates [Uysal, et al., Nature 389:610-614 (1997)]. Other components of the immune system, which are produced by adipose cells, include the protein adipsin, which is an integral part of the alternative complement system, and functions identically to human complement factor D [Rosen, B. S. et al., Science 244:1483-7 (1989)].
Little information is known about the role of the immune system as a mediator of obesity, but several recent studies suggest that the immune system may have an important contributory role in the development of obesity. Several cytokines are known to act as adipose tissue regulators. TNF-β suppresses the expression of β3 adreno-receptors on adipose cells, which are involved in sympathetically mediated lipolysis, while IL1 stimulates adipose leptin secretion [Sarraf, et al., Journal of experimental medicine 185:171-175 (1997)]. The metabolic activity rate of adipose cells has been observed to be closely correlated to their distance from the closest lymph node [Pond, C. M. et al., Proceedings of the nutrition society 60:365-374 (2001)], through a mechanism which is partly mediated by IL4, IL6 and TNF-β [Mattacks, C. A. et al., Cytokine 11:334-346 (1999)].
These observations, which point to the fact that obese animals and humans may also be suffering of various alterations in the different arms of the immune system, suggest that modulation of the immune system may change some of the pathogenic mechanisms responsible for the development of morbid obesity.
To the best of the inventor's knowledge, previously employed methods of immune modulation have not involved exposure of components of the immune system to mammalian naturally occurring β-glycolipids and specifically to a mixture of β-glycolipids.
WO 2005/032462, which is a previous publication by the present inventors, discloses the general use of intermediary metabolites and preferably, glucocerebrosides, in the treatment of immune-related disorders. The present invention now clearly shows that certain intermediary metabolites, the β-glycolipids and not the α-glycolipids are particularly effective, and specifically, β-lactosyl-ceramide (LacC), β-glucosylceramide (GluC), and β-galactosyl-ceramide (GalC)] and ceramide. Surprisingly, the inventors have now showed for the first time that β-lactosyl-ceramide may be used as a preferred β-glycolipid for immune-modulation.
Moreover, the inventors show a clear synergistic effect of a particular combination of two β-glycolipids, preferably a mixture of β-lactosyl-ceramide with β-glucosylceramide, which may be used as a powerful medicament for the treatment of immune-related disorders.
These and other objects of the invention will become clearer as the description proceeds